Much of the efficacy of allogeneic stem cell transplantation in treating hematologic malignancies is due to the T cell-mediated graft-vs-leukemia (GVL) effect. Chronic phase CML (CP-CML) is the most GVL-sensitive leukemia as demonstrated by a 5-fold increased risk of relapse in recipients of T cell-depleted grafts and a nearly 80% complete response rate in recipients of donor leukocyte infusions. Unfortunately, most other neoplasms are relatively GVL-resistant. The basis for differential susceptibility, even between such closely related leukemias as CP-CML and GVL-resistant blast crisis CML (BC-CML) is unknown. A detailed understanding of the mechanisms underlying GVL-resistance is the key first step in developing clinical approaches to make GVL-resistant cancers more GVL-sensitive. An obstacle in achieving this goal has been the absence of mouse models for GVL-sensitive and -resistant leukemias that share etiology and phenotype with their human counterparts. To address this we have been dissecting GVL responses against mouse models of GVL-sensitive chronic phase and GVL-resistant blast crisis CML. Mouse chronic phase CML (mCP-CML) is induced by the retroviral transduction of mouse bone marrow with bcr-abl. Mouse blast crisis CML (mBC-CML) is created by transducing bone marrow with both bcr-abl and a fusion between NUP98 and HOXA9 (NH). bcr-abl is the defining molecular abnormality in CML and NH is a "second hit" found in BC-CML. That leukemias are generated with retrovirus has allowed the creation of gene-deficient leukemias which are used to study GVL effector mechanisms. In sum, the basic mechanisms of T cell killing against mCP and mBC-CML are shared: cognate interactions between T cells and leukemias are required and impairment of any single effector mechanism does not diminish GVL. Nonetheless, in each case mBC-CML is relatively GVL-resistant. In order to better understand GVL-resistance, we have been working to identify the leukemia stem cells (LSC) for both mCP-CML and mBC-CML. The central hypothesis of this proposal is that LSCs are the key targets of GVL and that GVL resistance occurs in the interaction between effector T cells and the mBC-CML LSC. We will identify and purify mBC-CML and mCP-CML LSCs (Aim 1) and characterize them by flow cytometry and gene expression profiling to identify potential molecules that might promote GVL-resistance or sensitivity (Aim 2). We will develop novel in vitro and in vivo CTL assays against LSCs in Aim 3 using a method that allows a comparison of the sensitivity of mBC and mCP LSCs that express defined levels of the target peptide:MHCI complex. In Aim 4 we will create additional genetically-modified leukemias that underexpress or overexpress candidate mediators of GVL-resistance or -sensitivity (discovered in Aim 2). We will then test these leukemias in GVL experiments and their LSCs in the CTL assays developed in Aim 3. In this way we will have established a system for both identifying and validating candidate pathways for manipulation in clinical studies, which is our long-term objective. PUBLIC HEALTH RELEVANCE: Many people die from cancers of blood cells. Frequently immune cells, given as part of a bone marrow transplant from another person, are used to try to cure these patients. Unfortunately, for reasons that are unknown, many cancers are resistant to this therapy and this proposal will investigate the biology underlying this differential sensitivity.